IGEM:UBC/2009/Notebook/UBC iGEM 2010/2010/07/12

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dspB Track

Marianne and Vicki

PCR genomic preps (PCR I)

Start from the beginning again with the A. pleurpneumoniae strains. PCR out dspB from the strains, dilute product, PCR dspB again, extract dspB from gel (purification).

Strains:

  • H49: 80ng/uL x2
  • H80: 80ng/uL x2
  • H171: 60ng/uL x2
  • Total: 6 samples + 1 water control + 2 extra = 9 samples

Protocol: Common protocols for PCR

PCR Master mix
Reagent1x rxn volume (uL)Master Mix
10x rxn buffer2.5x922.5
10uM FW primer2x918
10uM RE primer2x918
10mM dNTP3x927
sdH2O10.3x992.7
Taq polymerase0.2x91.8
DNA5x945
Total25225

2 Master Mixes:

  • MMA (master mix A): His-tagged FW primer - 6 samples + 1 H2O water + 2 extra = 9 samples
  • MMB (master mix B): FW primer - 6 samples + 2 extra = 8 samples
  • Total 17 samples in all for master mix (13 PCR tubes in total - 12 samples & 1 H2O control)
PCR Tubes
LabelContent
1His-tagged H49 A
2His-tagged H49 B
3His-tagged H80 A
4His-tagged H80 B
5His-tagged H171 A
6His-tagged H171 B
7H49 A
8H49 B
9H80 A
10H80 B
11H171 A
12H171 B
13H2O control

PCR Cycles:

  • 95C @ 2 min
  • Cycle 30x:
    • 95C @ 30 sec
    • 65C @ 10 sec
    • 72C @ 80 sec
    • 72C @ 10 min
  • 10C @ hold

Start: 1316
End: 1446

Gel Verification

Protocol: gel verification protocol in iGEM training manual Gel orientation:

Gel orientation
123456100bp ladder78910111213

*Note: the second layer are Jason's samples - he is testing whether his PCR from last day worked or not

Machine conditions: 110V, 45 min, 0.5X TBE Buffer
Results:

Vicki Ma 23:00, 12 July 2010 (EDT)


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